We are working to develop flow-based methods for separating different cell types from a mixed-cell co-culture. We are currently pursuing two separate projects. In the first, we are co-culturing normal fibroblasts with transformed fibroblasts in order to determine if cell-cell interactions can overcome the growth inhibition which the normal cells experience in 3-D culture. In collaboration with Dr. Wright, we have developed subclones of each fibroblast cell type which are stably transfected with green fluorescent protein (GFP). We have characterized the stability of GFP expression in these cells using flow analysis, and are currently conducting experiments using simultaneous flow measurement of GFP and DNA content in order to measure the proliferative status of each cell type in a mixed-cell co-culture. The second project involves investigating the radiation response of monolayer and spheroid co-cultures of normal human prostate and fibroblast cells. We are inv esti gating several potential methods for using flow analysis and sorting to separate these cell types, including fuorescently-tagged antibodies directed against the cell surface, membrane labeling using PKH-dyes, and GFP transfection. New antibodies will be developed using phage display technology. Flow analysis will be critical in evaluating these methods and in isolating high-affinity antibodies for physical separation of the cells from a mixed-cell co-culture by the use of magnetic beads.